In general, the invention features screening methods involving nucleic acid-protein fusions.
Screening is considered to be an efficient tool to identify binding interactions between proteins and small molecule compounds derived from large pharmaceutically-based collections, new synthetic approaches such as combinatorial chemistry, or natural sources (TIBTECH, vol. 13, p. 115, 1995). However, the multidisciplinary nature of most screening techniques poses significant challenges. The most important challenge of such techniques is maintaining a ready supply of materials for the screen. Screening of small compound libraries with different protein targets requires sufficient amounts of compound. Alternatively, screening of large compound libraries (for example, having 106 members or greater) requires large amounts of recombinant protein. Another challenge is to operate the screen rapidly and cost effectively. Screening of compound libraries with different protein targets is generally time consuming if carried out in a sequential fashion.
Lately, a method has been described for the isolation of proteins with desired properties out of a pool of proteins (Szostak et al., Selection of Proteins Using RNA-Protein Fusions, U.S. Ser. No. 09/007,005, Jan. 14, 1998, now U.S. Pat. No. 6,258,558 B1, and U.S. Ser. No. 09/247,190, Feb. 9, 1999, now U.S. Pat. No. 6,261,804 B1; and Roberts and Szostak, Proc. Natl. Acad. Sci. USA (1997) vol. 94, p. 12297-12302). This technique is accomplished by means of protein-RNA fusion molecules where each protein is covalently linked to its encoding RNA. The protein-RNA fusion technology may be used to screen cDNA libraries and to clone new genes on the basis of protein-protein interactions (see, for example, Szostak et al., Selection of Proteins Using RNA-Protein Fusions, U.S. Ser. No. 09/007,005, Jan. 14, 1998, now U.S. Pat. No. 6,258,558 B1, and U.S. Ser. No. 09/247,190, Feb. 9, 1999, now U.S. Pat. No. 6,261,804 B1).
The purpose of the present invention is to efficiently identify protein-compound binding interactions (and, particularly, protein-small molecule interactions) by screening small molecule compounds with libraries of protein-nucleic acid fusions (for example, protein-RNA fusions) in a parallel fashion, thus providing a catalogue of small molecule-protein pairs.
Accordingly, in a first aspect, the invention features a method for detecting a compound-protein interaction, the method involving: (a) providing a compound library in which each member of the compound library is immobilized on a solid support; (b) contacting each member of the immobilized compound library in a single reaction chamber with each member of a protein-nucleic acid fusion library under conditions which allow the formation of compound-fusion complexes; (c) isolating the immobilized compound-fusion complexes; and (d) detecting the compound-fusion complex as an indication that the protein of the fusion interacts with the compound.
In preferred embodiments, the protein-nucleic acid fusion is either a protein-RNA fusion, a protein-DNA fusion, or a protein fused to a DNA-RNA hybrid; the solid support is a bead; each bead is coded with a unique detectable label; the compound of the complexed protein-nucleic acid fusion is identified by the unique detectable label associated with the bead; the detectable label is a peptide label, a nucleic acid label, a chemical label, a fluorescent label, or a radio frequency tag; the solid support is a chip and the compound library is immobilized on the chip in an addressable array; each member of the protein-nucleic acid fusion library is detectably labeled; the compound-fusion complex, or the components thereof, are recovered by release from the solid support; the method further involves recovering the protein-nucleic acid fusion from the solid support and identifying the protein; the identity of the protein is determined from the sequence of the nucleic acid portion of the protein-nucleic acid fusion; and the compound is a small molecule.
In a related aspect, the invention features a method for detecting a compound-protein interaction, the method involving: (a) providing a compound immobilized on a solid support; (b) contacting the immobilized compound with a protein-nucleic acid fusion library under conditions which allow the fusion to bind to the compound; and (c) detecting a bound protein-nucleic acid fusion as an indication that the protein of the protein-nucleic acid fusion interacts with the compound.
In preferred embodiments, the protein-nucleic acid fusion is either a protein-RNA fusion, a protein-DNA fusion, or a protein fused to a DNA-RNA hybrid; the protein-nucleic acid fusion is detectably labeled and the interaction is indicated by the association of the detectable label with the solid support; the bound protein-nucleic acid fusion is recovered by release from the solid support; the method further involves recovering the protein-nucleic acid fusion from the solid support and identifying the protein; the identity of the protein is determined from the sequence of the nucleic acid portion of the protein-nucleic acid fusion; the solid support is a column, glass slide, chip, or bead; and the compound is a small molecule.
As used herein, by a xe2x80x9clibraryxe2x80x9d is meant a collection of at least two molecules (for example, molecules such as compounds or protein-nucleic acid fusions). A compound library preferably includes at least 102 or 103 members, and, more preferably, at least 104, 105, or 106 members. A protein-nucleic acid library (for example, a protein-RNA library) preferably includes at least 102 or 103 members, more preferably, at least 104, 105, or 106 members, and, most preferably, at least 1010 or 1012 members.
By a xe2x80x9cDNA-RNA hybridxe2x80x9d is meant a DNA strand hybridized to a complementary RNA strand. Typically, the DNA strand is generated by reverse transcription of the RNA molecule.
By xe2x80x9caddressable arrayxe2x80x9d is meant a fixed pattern of immobilized objects on a solid surface in which the identity of the objects is known or can be readily determined.
By a xe2x80x9csmall moleculexe2x80x9d is meant a compound with a molecular weight of less than or equal to 10,000 Daltons, preferably, less than or equal to 1000 Daltons, and, most preferably, less than or equal to 500 Daltons.
The present invention provides a number of advantages. For example, the present methods reduce the amount of material required for a screen. In standard screens, considerable amounts of protein and small molecule compounds are required because each compound is screened with a single protein in a spatially segregated chamber. A library of protein-nucleic acid fusion molecules, however, can be screened for binding interactions with small molecule compounds in the same reaction chamber in a parallel fashion. In addition, the protein target need not be cloned, overexpressed, or isolated, but rather is screened as a protein-nucleic acid fusion molecule and identified by its coding nucleic acid. Moreover, material costs may be further reduced by miniaturization, which is facilitated by the present methods and is limited solely by the choice of detection method for the identification of small molecule-fusion complexes.
In addition, the present invention provides advantages in terms of the time required to carry out a compound screen. In particular, the methods described herein accelerate the identification of ligands (for example, small molecule ligands) by screening a library of protein targets with a library of potential ligands in a parallel fashion. In contrast to standard screens, where a small compound library is screened for binding to different proteins in a sequential fashion, small molecule compounds may be screened, in the present techniques, with a library of protein-nucleic acid fusions in a single assay. Consequently, the present invention facilitates the screening of members of a library of small molecule compounds for binding to the members of a library of proteins in a highly efficient manner.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.